Sunday, May 19, 2024
5/19/2024
Abstract Bacterial persistence contributes to antibiotic treatment failure and the relapse of many recalcitrant infections. Persisters are a subpopulation of transiently non-growing bacteria capable of surviving antimicrobial attacks from antibiotics and the immune system and eventually resuming growth. Many pathogens including, Salmonella enterica, Mycobacterium tuberculosis, and Staphylococcus aureus, form persisters within macrophages where they survive extended periods of time. It was shown that, although non-growing, Salmonella persisters retain the ability to express and inject effector proteins in macrophages leading to interference with the host immune response and supporting persister survival. Nonetheless, persisters remain vulnerable to macrophage-induced DNA damage in the form of double stranded breaks (DSBs) and require DSB repair through homologous recombination. Strikingly, intramacrophage Salmonella persisters also actively replicate chromosomal DNA and can accumulate more than four chromosome equivalents of DNA. I have found that persisters replicate complete chromosomes despite growth arrest and that this chromosome amplification is associated with a higher frequency of persister regrowth. I hypothesize that stresses encountered upon macrophage entry trigger a specific state of growth arrest where atypical chromosome replication is enabled and then favors repair of chromosomal DSBs by homologous recombination. To evaluate this hypothesis, I will decipher the mechanisms and consequences of DNA synthesis in Salmonella persisters. In Aim 1, I will characterize the contribution of chromosome amplification to homologous recombination and thus persister survival. I will use gene conversion assays to measure homologous recombination in persisters with high DNA content (1.1). I will then assess how DSB repair affects persister regrowth by tracking DSB repair using fluorescent imaging and transcriptional reporters of the DNA damage response (1.2). In Aim 2, I will determine the basis for DNA synthesis despite growth arrest including the requirements for initiation of DNA synthesis and intramacrophage conditions that trigger this atypical DNA synthesis. I will determine the requirements for initiation of chromosome replication at oriC through minichromosome replication assays (2.1). I will assess the macrophage triggers for atypical DNA replication by evaluating DNA accumulation of persisters in genetically- altered macrophages (2.2). Altogether, this research will further our understanding of intracellular persisters including formation, maintenance of the persistent state, and re-growth. Mechanistic understanding of persister survival will ultimately contribute to the development of approaches for targeting persisters, enhancing antibiotic efficacy, and preventing the development of antibiotic resistance.
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